All bacteria rely on mRNA degradation as a means to regulate gene expression and to adapt to the ever changing milieu in which they live. For a number of transcripts, rapid degradation is governed by the phosphorylation state of the 5' end, which can potentiate RNA decay by nearly an order of magnitude if monophosphorylated. RppH was recently identified as the enzyme responsible for triggering 5' end-dependent degradation by removing phosphates from the 5' end of primary transcripts to produce short-lived 5' monophosphorylated decay intermediates; however, our knowledge of the role of RppH in the larger context of cellular RNA metabolism remains quite rudimentary. The primary purpose of this proposal is to further our understanding of 5' end-dependent RNA decay in bacteria, using Escherichia coli and Bacillus subtilis as model organisms. To achieve this goal, a reporter system will be created to detect changes in 5' end- dependent mRNA degradation, and forward genetics will be used to: 1. identify additional sources of pyrophosphohydrolase activity; and 2. discover positive and negative regulators of RppH. Promising candidates will be further characterized by biochemical and molecular biological assays to determine the scope and mechanism of their influence on 5' end-dependent RNA decay. The results of these studies will enhance our understanding of how bacteria use RNA degradation to control their gene expression profile and provide insights into a regulatory process known to influence bacterial pathogenesis. PUBLIC HEALTH RELEVANCE: RppH is a bacterial protein previously implicated as being important for pathogens to invade human cells. Furthermore, all bacteria-non-pathogenic and pathogenic alike-rely on mRNA degradation to regulate gene expression and to adapt to their surroundings. These studies will enhance and deepen our understanding of a fundamental cellular process, thereby providing insight into a molecular mechanism known to influence bacterial pathogenesis.